Methylene Blue Stain

In this process, bacteria are placed on a slide, heat-killed and stained with a basic dye called methylene blue. This stain makes it easier to inspect the morphology, or shape, of the bacteria. Some bacteria are rod-shaped, while others are spherical or even spiral; methylene blue staining helps microbiologists recognize these structural differences.

There are two ways to prepare a specimen to be observed under a light com­pound microscope. These are a wet mount and a smear.

A wet mount is a preparation process where a live specimen in culture fluid is placed on a concave glass side or a plain glass slide. The microorganism is free to move about within the fluid, although the viscosity of the substance slows its movement. This makes it easier for you to observe the microorganism. The specimen and the substance are protected from spillage and outside contaminates by a glass cover that is placed over the concave portion of the slide.

A smear is a preparation process where a specimen that is spread on a slide. You prepare a smear using the heat fixation process. For most staining procedures one need only air dry the film by holding the slide high above a bunsen flame, when dry pass the slide film side up, three times through the flame to kill and fix the cells. Too much heat will distort the shape of the organism and could alter its uptake of stain - the slide should feel warm but not hot. Bear in mind that staining procedures kill the cells and that some of the cells characteristics may inevitably be altered. All stainings are to be carried out over the staining rack or in the sinks.

Prepare a stained vital "crushed drop"specimen of agar microbial culture.

PROCEDURE:

1. Obtain a clean microscope slide.

2. Place one drop of water on the slide.

3. Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony.

4. Gently mix the bacteria into the water drop.

5. Place one drop of a weak solution (1:1000) dye (methylene dark blue or fuchsin).

6. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of specimen.

7. Slowly lower the coverslip into place. 

8. View the microorganism with the high-power (x40) objective. 

9. Sketch a picture of the microorganism. 

10. Sign the picture and specify Total Magnification (TM).

Prepare a stained fixed smear of agar microbial culture.Heat Fixing the Microbial Sample

Before staining, the sample must be heat fixed. This process accomplishes three things. It functions to:

• kill the bacteria

• firmly affix the smear to the microscope slide

• allow the sample to more readily take up the stain.

In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air dry. Then either place the slide in the slide holder of a microincinerator, or pass the dried slide through the flame of a Bunsen burner 3 or 4 times, smear side facing up. Once the slide is heat fixed, it can then be stained.

PROCEDURE:

You prepare a smear using the heat fixation process:

1. Use a clean glass slide.

2. Take a loop of the culture.

3. Place the live microorganism on the glass slide.

4. The slide is air dried then passed over a Bunsen burner about three times. The heat causes the microorganism to adhere to the glass slide. This is known as fixing the microorganism to the glass slide.

Stain the microorganism with an appropriate stain: